The recent discovery of iPSC (induced Pluripotent Stem Cell) technology marks a promising breakthrough in regenerative medicine. The beauty of the technology is its ability to convert adult mature cells into embryonic stem cells through the expression of a cocktail of essential factor genes. Thus, iPSCs bypass the ethical dilemma of using embryonic materials and eggs. In addition, the creation of iPSCs for individual patients using their own cells can avoid immune rejection and achieve successful therapeutic effects. Since its initial discovery, the method has been used to generate patient-specific stem cells for regenerative therapy and drug screening, including Parkinson disease, sickle cell anemia, Huntington disease and many other genetic diseases. It is predicted that patients may someday be treated with their own healthy versions of stem cells.
The technology of iPSC induction, however, is in its infancy. Generation of iPS cells depends on the synthesis of factor proteins that regulate the developmental clock of adult cells in order to return them to the embryonic state. Viruses are a common approach to deliver factor genes into the cell but they incur the risks of gene mutation and instability. Most critically, the efficiency of iPSC induction is extremely low with all existing approaches. Without clearing these road blocks, it would be impossible to translate this technology to the clinic in the near future.
In this project, we propose to fundamentally improve this technology by re-engineering the iPS-inducing factors. We will modify part of these factor proteins, which function inefficiently, to become highly potent in activating target genes related to stemness. By functional screening, we will identify the most potent set of factors for iPSC induction. In addition, we have identified a novel iPS-inducing factor in our lab. Together with those engineered factors, we will work out an ideal cocktail of factors that robustly induce iPSCs. To make virus-free iPS cells, we have developed an enzyme that specifically recognizes and removes the virus-delivered factor genes in the host cell. The removal of the viral transgene will avoid tumor formation and increase clinical safety. Finally, we will generate the safest, genetic material-free stem cells directly by using proteins produced by the genes of engineered factors.
With these approaches, the generation of iPSCs will be much more robust, enabling us to create patient-specific stem cells efficiently and safely within a short period of time. We will be the first to break the current technical bottleneck by modifying their protein structure. Taken together, this project may ultimately revolutionize the existing ways to create genetically tailored stem cell lines for research and disease treatment in regenerative medicine.

Statement of Benefit to California:

Induced pluripotent stem cells (iPSCs) offer hope and promise for the therapeutic usage of personalized stem cell in regenerative medicine. Among ten leading death causes in California, five of them can directly benefit from cell-based tissue regeneration, including heart disease, stroke, Alzheimer’s disease, diabetes, and liver diseases. Currently, the economic burdens derived from these diseases are enormous. It is estimated from State of California, Department of Public Health that California taxpayers pay 48 billion annually for cardiovascular diseases, 73 billion excluding non-paid family care for Alzheimer’s disease, and 116 billion for diabetes-related diseases.
By far, personated iPSC lines have been successfully made from patients with a variety of diseases, including Alzheimer’s disease, Parkinson disease, sickle cell anemia, Huntington disease, muscular dystrophy and many other genetic diseases. It is hoped that these patients may someday be treated with their own healthy versions of stem cells. However, the iPSC technology still has several shortcomings inhibiting its clinical application. Our proposed research aims at improving this technology by revolutionizing existing methods in producing iPSCs and freeing them from cancer-causing side-effects. This research will finally lead to a new way of developing personalized stem cells for therapy and possibly a cure to above mentioned diseases.
The goal of this study is to robustly produce iPSCs that are safe for therapeutic applications. It is thus in line with the mission of the California Institute for Regenerative Medicine – to promote rapid progress in stem cell research leading to treatments and cures for diseases and to the growth of a stem cell and regenerative medicine industry critical to future clinical applications.
Our proposed improvements in the production of iPSCs, when translated into therapeutic interventions, will directly benefit the health of California citizens and reduce the economic burden presently borne by California taxpayers. Indirectly, we believe this research will increase California’s visibility in stem cells research, attract more federal funding to sponsor future research and fulfill the wish of California citizens who voted to support stem cell research. It may also stimulate California’s economy growth by stimulating the iPSC regenerative medicine industry for the treatment or cure of diseases.

Review Summary:

The applicants propose to overcome the technical bottleneck of inefficient production of induced pluripotent stem cells (iPSCs) using the currently available sets of transcription factors delivered by a retroviral vector. Their working hypothesis is that increasing the factors’ transactivation capacity on target genes will strongly enhance the efficiency of reprogramming to yield iPSCs. To accomplish this goal, the applicants will first engineer defined factors with modified transactivation domains. They will then test and determine the most effective combination of engineered factors to generate iPSCs. The applicants have also devised a modified enzyme that will be utilized for efficient removal of the integrated viral transgenes. Finally, the applicants propose to produce the engineered factors as purified recombinant proteins that will be directly introduced to target cells, avoiding introduction of any genetic materials.
If successful, reviewers felt that the applicants could improve the technology quantitatively, potentially by several orders of magnitude. The approach to engineering the transcription factors was considered innovative. A minor weakness, however, is that the applicants do not consider synergies with advances that already have been made in the use of small molecules to promote reprogramming. They also propose to switch from use of a viral vector for delivery of the modified transcription factor to production of the corresponding proteins. This portion of the proposal was less well described and less innovative as it is derivative of work already published by other groups. Overall, the molecular biology proposed for construction of the engineered transcription factors was thought to be innovative and potentially of high impact.
The applicants present impressive preliminary data supporting the feasibility of the approach and supporting the high quality of their experimental design. The principle goal of generating higher activity forms of the transcription factors, showing that these turn on endogenous genes and that they contribute to production of reprogrammed cells in which relevant genes associate in a common active chromatin territory, is documented for one key transcription factor. The applicants present a logical and convincing timeline for the production and testing of the engineered genes and delivery system. The proposal to switch over to recombinant factor proteins for iPSC production was less compelling since the approach may not add much value if high efficiency reprogramming and subsequent excision of the reprogramming viral vector can be achieved. Reviewers identified some areas that should have been addressed in the plan including possible pitfalls in refolding and use of recombinant proteins, as this may not be straightforward. The possible use of small molecules already identified to contribute to reprogramming was also not discussed. Reviewers also felt that the assessment of the biology of iPSC generated as a function of the reprogramming factors was not fully addressed. For example, the extent of cellular plasticity, stability of the reprogrammed phenotype, or completeness of epigenetic change were not discussed in depth in the application.
The principal investigator (PI) has worked extensively with iPSCs, has noted strengths in molecular biology, and is well qualified to perform the described experiments. The PI will contribute 36% effort on the project. Additional team members who will contribute the most hands-on time on the project appear qualified. Reviewers noted a need for additional experience with human ES and iPSC biology to help assess the consequences of genetic manipulations to the overall cell physiology.

Programmatic review:

A motion was made to move this application into Tier 1, Recommended for Funding. The GWG felt that the proposal could have a broad impact and also felt that contributions from industry could lead to commercialization and should be encouraged. The motion carried.